Biosensor, iontophoretic sampling system, and methods of use thereof

ABSTRACT

An automated system for continual transdermal extraction of analytes present in a biological system is provided. The system can be used for detecting and/or measuring the concentration of the analyte using an electrochemical biosensor detection means. The system optionally uses reverse iontophoresis to carry out the continual transdermal extraction of the analytes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 09/174,902, filed Oct. 19, 1998, now abandoned, from whichpriority is claimed under 35 USC §120, which is related to provisionalpatent applications Ser. Nos. 60/077,993, filed Mar. 13, 1998, and60/080,591, filed Apr. 3, 1998, from which priority is claimed under 35USC §119(e)(1), and which applications are incorporated herein byreference in their entireties.

TECHNICAL FIELD

This invention relates generally to a biosensor for use in monitoringthe concentration of target chemical analytes present in an aqueousbiological system. More particularly, the invention relates to abiosensor for measuring the concentration of one or more analytes in atransdermally extracted sample. The invention also relates to anelectrode system for continual transdermal extraction of one or moreanalytes from a biological system over an extended period of operation.One important application of the invention involves a sampling systemfor monitoring blood glucose using noninvasive or minimally invasivesampling techniques.

BACKGROUND

A number of diagnostic tests are routinely performed on humans toevaluate the amount or existence of substances present in blood or otherbody fluids. These diagnostic tests typically rely on physiologicalfluid samples removed from a subject, either using a syringe or bypricking the skin. One particular diagnostic test entailsself-monitoring of blood glucose levels by diabetics.

Diabetes is a major health concern, and treatment of the more severeform of the condition, Type I (insulin-dependent) diabetes, requires oneor more insulin injections per day. Insulin controls utilization ofglucose or sugar in the blood and prevents hyperglycemia which, if leftuncorrected, can lead to ketosis. On the other hand, improperadministration of insulin therapy can result in hypoglycemic episodes,which can cause coma and death. Hyperglycemia in diabetics has beencorrelated with several long-term effects of diabetes, such as heartdisease, atherosclerosis, blindness, stroke, hypertension and kidneyfailure.

The value of frequent monitoring of blood glucose as a means to avoid orat least minimize the complications of Type I diabetes is wellestablished. Patients with Type II (non-insulin-dependent) diabetes canalso benefit from blood glucose monitoring in the control of theircondition by way of diet and exercise.

Conventional blood glucose monitoring methods generally require thedrawing of a blood sample (e.g., by fingerprick) for each test, and adetermination of the glucose level using an instrument that readsglucose concentrations by electrochemical or calorimetric methods. TypeI diabetics must obtain several fingerprick blood glucose measurementseach day in order to maintain tight glycemic control. However, the painand inconvenience associated with this blood sampling, along with thefear of hypoglycemia, has lead to poor patient compliance, despitestrong evidence that tight control dramatically reduces long-termdiabetic complications. In fact, these considerations can often lead toan abatement of the monitoring process by the diabetic.

Recently, various methods for determining the concentration of bloodanalytes without drawing blood have been developed. For example, U.S.Pat. No. 5,267,152 to Yang et al. describes a noninvasive technique ofmeasuring blood glucose concentration using near-IR radiationdiffuse-reflection laser spectroscopy. Similar near-IR spectrometricdevices are also described in U.S. Pat. No. 5,086,229 to Rosenthal etal. and U.S. Pat. No. 4,975,581 to Robinson et al.

U.S. Pat. No. 5,139,023 to Stanley describes a transdermal blood glucosemonitoring apparatus that relies on a permeability enhancer (e.g., abile salt) to facilitate transdermal movement of glucose along aconcentration gradient established between interstitial fluid and areceiving medium. U.S. Pat. No. 5,036,861 to Sembrowich describes apassive glucose monitor that collects perspiration through a skin patch,where a cholinergic agent is used to stimulate perspiration secretionfrom the eccrine sweat gland. Similar perspiration collection devicesare described in U.S. Pat. No. 5,076,273 to Schoendorfer and U.S. Pat.No. 5,140,985 to Schroeder.

In addition, U.S. Pat. No. 5,279,543 to Glikfeld describes the use ofiontophoresis to noninvasively sample a substance through skin into areceptacle on the skin surface. Glikfeld suggests that this samplingprocedure can be coupled with a glucose-specific biosensor orglucose-specific electrodes in order to monitor blood-glucose. Finally,International Publication No. WO 96/00110 to Tamada describes aniontophoretic apparatus for transdermal monitoring of a targetsubstance, where an iontophoretic electrode is used to move an analyteinto a collection reservoir and a biosensor is used to detect the targetanalyte present in the reservoir.

However, there remains a need in the art for sampling devices andsampling methods which provide low cost, accurate determination ofanalyte concentrations in field or home-testing applications,particularly where continual and/or automatic monitoring is desired.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides an efficient sampling systemfor detecting and/or measuring the concentration of a transdermallyextracted analyte. The invention represents an improvement over priornoninvasive monitoring techniques and devices by providing an automaticsampling system coupled with a highly sensitive biosensor fordetermining the concentration of a target analyte present in an aqueousbiological system. The sampling system extracts small amounts of atarget analyte via transdermal methods, and then senses and/orquantifies the concentration of the target analyte. Sampling is carriedout in a continual manner, allowing quantification to be carried outeven when a target analyte, extracted from the biological system, isobtained at a sub-millimolar (sub-mM) concentration.

The advantages provided by the invention are thus several fold. Forexample, the noninvasive nature of the sampling system significantlyincreases the likelihood of patient acceptance. In the particularcontext of blood glucose monitoring, better glycemic control can beachieved by taking frequent blood glucose measurements on a daily basis,and using that information to determine the amount and frequency ofinsulin administration. Use of the noninvasive sampling system of theinvention helps increase the likelihood that such frequent measurementswill be taken. In addition, the automatic sampling provided by theinstant sampling system, particularly when taken over an extended periodof time (e.g., 24 hours or more) can be used to monitor concentrationswings previously not detectable using prior devices. Again in thecontext of blood glucose monitoring, it is now believed that even fourto seven glucose measurements per day may be insufficient to reflect thediurnal glucose level variation in many diabetics. Using the instantsampling system to automatically measure blood glucose at, for example,a frequency of once per hour, allows monitoring of previouslyunrecognizable glucose swings, particularly when a subject is asleep.Thus, the invention provides access to information that is of greatclinical benefit in home, field and/or medical environments.

Accordingly, it is a general object of the invention to provide anautomated system for continual transdermal extraction of analytespresent in biological fluids. In one particular embodiment, thetransdermal extraction is carried out using reverse iontophoresis orelectroosmosis to extract analytes across a subject's skin. In thisembodiment, one or more collection reservoirs are contacted with asubject's skin. The reservoirs typically contain a conductive medium andare in contact with a sampling means for providing electric potential orcurrent between the reservoir site and another site on the subject'sskin. A biosensor is also in contact with the one or more reservoirs,and provides a means for sensing and/or quantifying the concentration ofa target analyte present in the reservoirs.

In a preferred embodiment, an automated system for iontophoreticextraction of analytes is provided, wherein iontophoretic electrodescapable of continual cycling under iontophoretic conditions are used totransdermally extract analytes continually over a period of about 1-24hours, or longer. Therefore, unlike most iontophoresis applications, theiontophoretic electrodes of the invention are capable of passing currentin both directions without concomitantly participating in undesirableside reactions, particularly water hydrolysis. In addition, theelectrodes must have the capacity to pass a high amount of charge, whichcapacity is readily reversible so that the electrodes pass currentreproducibly for an extended period of operation.

In another embodiment, an automated system for continual transdermalextraction of analytes present in biological fluids is provided, whereinthe transdermal extraction is carried out using sonophoresis to extractanalytes across a subject's skin. In this embodiment, a collectionreservoir is contacted with a subject's skin. The reservoir contains aconductive medium, and is in contact with a sampling means for applyingultrasound to the contacted skin surface such that noninvasive samplingof analytes beneath the skin surface can be carried out. A biosensor isalso in contact with the reservoir, providing a means for sensing and/orquantifying the concentration of a target analyte extracted into thereservoir.

In each of the iontophoretic and sonophoretic sampling systems of theinvention, the collection reservoirs are comprised of a liquid, orliquid-containing medium which is ionically conductive and efficientlytransmits the electric potential or current, or the ultrasound, betweenthe respective sampling means and the skin surface. In preferredembodiments, the liquid-containing medium is an ionically conductivehydrogel or wicking material soaked with an ionically conductive medium.

As will be understood by the ordinarily skilled artisan upon reading thepresent specification, there are a large number of analytes that can besampled using the present automated sampling systems. In systems whichrely on the reverse iontophoresis/electroosmosis techniques describedherein, charged (e.g., having a negative or positive ionic charge)substances will be extracted at the highest concentrations, whileuncharged substances will be extracted at lower, albeit stillquantifiable, concentrations. One particular uncharged analyte ofinterest herein is glucose. Other analytes of interest include, but arenot limited to, amino acids, enzyme substrates or products indicative ofa disease state or condition, therapeutic agents, drugs of abuse, andelectrolytes.

The biosensor used for sensing and/or quantitating the target analyteextracted by the present sampling system needs to perform reliably andreproducibly using extracted concentrations (e.g., sub-mM) which arewell below those measured by conventional electrochemical detection(generally in the mM range). As used herein, “sub-mM” refers to andconcentration that is less than 1 mM. In one particular embodiment, thebiosensor includes an electrode comprising a platinum-group metal (e.g.,Pt, Pd, Ru, and Rh). The biosensor electrode is used to detect hydrogenperoxide generated by an enzyme oxidase which specifically reacts withan analyte of interest to provide hydrogen peroxide. Since the automaticsampling system is used to provide continual or periodic sampling overan extended period of operation, the biosensor electrode must have a lowbackground current, and be stable for at least about 1-24 hours ofoperation. The biosensor electrode further must have high sensitivityfor the hydrogen peroxide signal, where a preferred sensitivity(nA/μM):background current (nA) ratio is on the order of about 3 orgreater. Finally, the biosensor electrode must exhibit reduced catalyticperoxide decomposition by the platinum-group metal constituent.

Accordingly, it is a primary object of the invention to provide samplingsystem for monitoring the concentration of an analyte present in abiological system. The sampling system comprises:

(a) a reservoir containing an ionically conductive medium and an enzymecapable of reacting with the analyte to produce hydrogen peroxide;

(b) sampling means in operative contact with the reservoir, wherein thesampling means is used for extracting the analyte from the biologicalsystem into the reservoir to obtain a sub-millimolar (sub-mM)concentration of the analyte in the reservoir which reacts with theenzyme to produce hydrogen peroxide; and

(c) a sensor element also in operative contact with the reservoir,wherein the sensor element reacts electrochemically with the hydrogenperoxide produced in the reservoir to provide a detectable signal. Thesensor element comprises an electrode having suitable geometric surfacearea and background noise so as to be effective in the present samplingsystem.

It is also a primary object of the invention to provide a samplingsystem for monitoring the concentration of an analyte present in abiological system, wherein the sampling system comprises:

(a) a reservoir containing an ionically conductive medium and an enzymecapable of reacting with the analyte to produce hydrogen peroxide;

(b) reverse iontophoretic sampling means in operative contact with thereservoir, wherein the reverse iontophoretic sampling means is used forextracting the analyte from the biological system into the reservoir toobtain a sub-millimolar (sub-mM) concentration of the analyte in thereservoir which reacts with the enzyme to produce hydrogen peroxide; and

(c) a sensor element also in operative contact with the reservoir,wherein the sensor element reacts electrochemically with the hydrogenperoxide produced in the reservoir to provide a detectable signal. Thereverse iontophoretic sampling means comprises first and secondiontophoretic electrodes having suitable geometric area and currentcarrying capability so as to be operative in the present samplingsystem.

It is a still further object of the invention to provide a method formonitoring the concentration of an analyte present in a biologicalsystem, wherein the method comprises the following steps:

(a) extracting an analyte from the biological system into a collectionreservoir to provide a sub-millimolar (sub-mM) concentration of theanalyte in the reservoir;

(b) contacting the analyte extracted in step (a) with an enzyme thatreacts with the analyte to produce hydrogen peroxide;

(c) detecting the hydrogen peroxide produced in step (b) with a sensorelement, wherein the sensor element reacts electrochemically with thehydrogen peroxide to produce a detectable signal;

(d) measuring the signal produced in step (c);

(e) correlating the measurement obtained in step (d) with theconcentration of the analyte in the biological system; and

(f) performing steps (a)-(d) continually or periodically over anextended period of operation. The sensor element comprises an electrodehaving suitable geometric surface area and background noise so as to beoperative in the present method. Optionally, the method can be carriedout using a reverse iontophoretic system to transdermally extract theanalyte from the biological system, wherein the iontophoretic electrodeshave suitable geometric area and current carrying capability so as to beoperative in the present method.

In a further aspect of the above embodiments, the sensor element canalso include a reference electrode, and a counter electrode. Further, acounter electrode of the sensor element and an iontophoretic electrodeof the sampling system can be combined as a single bimodal electrodewhere the electrode is not used simultaneously for both functions, i.e.,where the counter and iontophoretic functions are separately carried outat different times.

Additional objects, advantages and novel features of the invention willbe set forth in part in the description which follows, and in part willbecome apparent to those skilled in the art upon examination of thefollowing, or may be learned by practice of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the reaction which glucoseoxidase (GOx) catalyzes to obtain gluconic acid and hydrogen peroxide,and result in the generation of a current.

FIG. 2 is an exploded pictorial representation of components from apreferred embodiment of the automatic sampling system of the presentinvention.

FIG. 3 is a representation of one embodiment of a bimodal electrodedesign. The figure presents an overhead and schematic view of theelectrode assembly 33. In the figure, the bimodal electrode is shown atand can be, for example, a Ag/AgCl iontophoretic/counter electrode. Thesensing or working electrode (made from, for example, platinum) is shownat 31. The reference electrode is shown at 32 and can be, for example, aAg/AgCl electrode. The components are mounted on a suitable substance34. In this example of such an electrode the working electrode area isapproximately 1.35 cm².

FIG. 4 is a representation of a cross-sectional schematic view of thebimodal electrodes as they may be used in conjunction with a referenceelectrode and a hydrogel patch. In the figure, the components are asfollows: bimodal electrodes 40 and 41; sensing electrodes 42 and 43;reference electrodes 44 and 45; a substrate 46; and hydrogel pads 47 and48.

DETAILED DESCRIPTION OF THE INVENTION

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particular compositionsor biological systems as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a”, “an” and “the” include plural referentsunless the content clearly dictates otherwise. Thus, for example,reference to “a binder” includes a mixture of two or more such binders,reference to “an analyte” includes mixtures of analytes, and the like.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice for testing of the present invention, the preferredmaterials and methods are described herein.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions set outbelow.

The terms “analyte” and “target analyte” are used herein to denote anyphysiological analyte of interest that is a specific substance orcomponent that is being detected and/or measured in a chemical,physical, enzymatic, or optical analysis. A detectable signal (e.g., achemical signal or electrochemical signal) can be obtained, eitherdirectly or indirectly, from such an analyte. Furthermore, the terms“analyte” and “substance” are used interchangeably herein, and areintended to have the same meaning, and thus encompass any substance ofinterest. In preferred embodiments, the physiological analyte ofinterest is, for example, glucose, or a chemical that has aphysiological action, for example a drug or pharmacological agent.

A “sampling device” or “sampling system” refers to any device forobtaining a sample from a biological system for the purpose ofdetermining the concentration of an analyte of interest. As used herein,the term “sampling” means invasive, minimally invasive or non-invasiveextraction of a substance from the biological system, generally across amembrane such as skin or mucosa. The membrane can be natural orartificial, and can be of plant or animal nature, such as natural orartificial skin, blood vessel tissue, intestinal tissue, and the like.Typically, the sampling means are in operative contact with a“reservoir,” wherein the sampling means is used for extracting theanalyte from the biological system into the reservoir to obtain theanalyte in the reservoir. A “biological system” includes both living andartificially maintained systems. Examples of minimally invasive andnoninvasive sampling techniques include iontophoresis, sonophoresis,suction, electroporation, thermal poration, passive diffusion, microfine(miniature) lances or cannulas, subcutaneous implants or insertions, andlaser devices. Sonophoresis uses ultrasound to increase the permeabilityof the skin (see, e.g., Menon et al. (1994) Skin Pharmacology7:130-139). Suitable sonophoresis sampling systems are described inInternational Publication No. WO 91/12772, published Sep. 5, 1991.Passive diffusion sampling devices are described, for example, inInternational Publication Nos.: WO 97/38126 (published Oct.16, 1997); WO97/42888, WO 97/42886, WO 97/42885, and WO 97/42882 (all published Nov.20, 1997); and WO 97/43962 (published Nov. 27, 1997). Laser devices usea small laser beam to burn a hole through the upper layer of thepatient's skin (see, e.g., Jacques et al. (1978) J. Invest. Dermatology88:88-93). Examples of invasive sampling techniques include traditionalneedle and syringe or vacuum sample tube devices.

A “monitoring system,” as used herein, refers to a system useful forcontinually or continuously measuring a physiological analyte present ina biological system. Such a system typically includes, but is notlimited to, sampling means, sensing means, and a microprocessor means inoperative communication with the sampling means and the sensing means.

The term “artificial,” as used herein, refers to an aggregation of cellsof monolayer thickness or greater which are grown or cultured in vivo orin vitro, and which function as a tissue of an organism but are notactually derived, or excised, from a pre-existing source or host.

The term “subject” encompasses any warm-blooded animal, particularlyincluding a member of the class Mammalia such as, without limitation,humans and nonhuman primates such as chimpanzees and other apes andmonkey species; farm animals such as cattle, sheep, pigs, goats andhorses; domestic mammals such as dogs and cats; laboratory animalsincluding rodents such as mice, rats and guinea pigs, and the like. Theterm does not denote a particular age or sex. Thus, adult and newbornsubjects, as well as fetuses, whether male or female, are intended to becovered.

As used herein, the term “continual measurement” intends a series of twoor more measurements obtained from a particular biological system, whichmeasurements are obtained using a single device maintained in operativecontact with the biological system over the time period in which theseries of measurements is obtained. The term thus includes continuousmeasurements.

The term “transdermal,” as used herein, includes both transdermal andtransmucosal techniques, i.e., extraction of a target analyte acrossskin or mucosal tissue. Aspects of the invention which are describedherein in the context of “transdermal,” unless otherwise specified, aremeant to apply to both transdermal and transmucosal techniques.

The term “transdermal extraction,” or “transdermally extracted” intendsany noninvasive, or at least minimally invasive sampling method, whichentails extracting and/or transporting an analyte from beneath a tissuesurface across skin or mucosal tissue. The term thus includes extractionof an analyte using iontophoresis (reverse iontophoresis),electroosmosis, sonophoresis, microdialysis, suction, and passivediffusion. These methods can, of course, be coupled with application ofskin penetration enhancers or skin permeability enhancing technique suchas tape stripping or pricking with micro-needles. The term“transdermally extracted” also encompasses extraction techniques whichemploy thermal poration, electroporation, microfine lances, microfinecanulas, subcutaneous implants or insertions, and the like.

The term “iontophoresis” intends a method for transporting substancesacross tissue by way of an application of electrical energy to thetissue. In conventional iontophoresis, a reservoir is provided at thetissue surface to serve as a container of material to be transported.Iontophoresis can be carried out using standard methods known to thoseof skill in the art, for example by establishing an electrical potentialusing a direct current (DC) between fixed anode and cathode“iontophoretic electrodes,” alternating a direct current between anodeand cathode iontophoretic electrodes, or using a more complex waveformsuch as applying a current with alternating polarity (AP) betweeniontophoretic electrodes (so that each electrode is alternately an anodeor a cathode).

The term “reverse iontophoresis” refers to the movement of a substancefrom a biological fluid across a membrane by way of an applied electricpotential or current. In reverse iontophoresis, a reservoir is providedat the tissue surface to receive the extracted material.

“Electroosmosis” refers to the movement of a substance through amembrane by way of an electric field-induced convective flow. The termsiontophoresis, reverse iontophoresis, and electroosmosis, will be usedinterchangeably herein to refer to movement of any tonically charged oruncharged substance across a membrane (e.g., an epithelial membrane)upon.application of an electric potential to the membrane through anionically conductive medium.

The term “sensing device,” “sensing means,” or “biosensor device”encompasses any device that can be used to measure the concentration ofan analyte, or derivative thereof, of interest. Preferred sensingdevices for detecting blood analytes generally include electrochemicaldevices and chemical devices. Examples of electrochemical devicesinclude the Clark electrode system (see, e.g., Updike et al. (1967)Nature 214:986-988) and other amperometric, coulometric, orpotentiometric electrochemical devices. Examples of chemical devicesinclude conventional enzyme-based reactions as used in the Lifescan®glucose monitor (Johnson and Johnson, New Brunswick, N.J.) (see, e.g.,U.S. Pat. No. 4,935,346 to Phillips et al.).

A “biosensor” or “biosensor device” includes, but is not limited to, a“sensor element” which includes, but is not limited to, a “biosensorelectrode” or “sensing electrode” or “working electrode” which refers tothe electrode that is monitored to determine the amount of electricalsignal at a point in time or over a given time period, which signal isthen correlated with the concentration of a chemical compound. Thesensing electrode comprises a reactive surface which converts theanalyte, or a derivative thereof, to electrical signal. The reactivesurface can be comprised of any electrically conductive material suchas, but not limited to, platinum-group metals (including, platinum,palladium, rhodium, ruthenium, osmium, and iridium), nickel, copper,silver, and carbon, as well as, oxides, dioxides, combinations or alloysthereof. Some catalytic materials, membranes, and fabricationtechnologies suitable for the construction of amperometric biosensorswere described by Newman, J. D., et al. (Analytical Chemistry 67 (24),4594-4599, 1995).

The “sensor element” can include components in addition to a biosensorelectrode, for example, it can include a “reference electrode,” and a“counter electrode.” The term “reference electrode” is used herein tomean an electrode that provides a reference potential, e.g., a potentialcan be established between a reference electrode and a workingelectrode. The term “counter electrode” is used herein to mean anelectrode in an electrochemical circuit which acts as a current sourceor sink to complete the electrochemical circuit. Although it is notessential that a counter electrode be employed where a referenceelectrode is included in the circuit and the electrode is capable ofperforming the function of a counter electrode, it is preferred to haveseparate counter and reference electrodes because the referencepotential provided by the reference electrode is most stable when it isat equilibrium. If the reference electrode is required to act further asa counter electrode, the current flowing through the reference electrodemay disturb this equilibrium. Consequently, separate electrodesfunctioning as counter and reference electrodes are most preferred.

In one embodiment, the “counter electrode” of the “sensor element”comprises a “bimodal electrode.” The term “bimodal electrode” as usedherein typically refers to an electrode which is capable of functioningnon-simultaneously as, for example, both the counter electrode (of the“sensor element”) and the iontophoretic electrode (of the “samplingmeans”).

The terms “reactive surface,” and “reactive face” are usedinterchangeably herein to mean the surface of the sensing electrodethat: (1) is in contact with the surface of an electrolyte containingmaterial (e.g. gel) which contains an analyte or through which ananalyte, or a derivative thereof, flows from a source thereof; (2) iscomprised of a catalytic material (e.g., carbon, platinum palladium,rhodium, ruthenium, or nickel and/or oxides, dioxides and combinationsor alloys thereof) or a material that provides sites for electrochemicalreaction.; (3) converts a chemical signal (e.g. hydrogen peroxide) intoan electrical signal (e.g., an electrical current); and (4) defines theelectrode surface area that, when composed of a reactive material, issufficient to drive the electrochemical reaction at a rate sufficient togenerate a detectable, reproducibly measurable, electrical signal thatis correlatable with the amount of analyte present in the electrolyte.

The term “collection reservoir” is used to describe any suitablecontainment means for containing a sample extracted from a biologicalsystem. The reservoir can include a material which is ionicallyconductive (e.g., water with ions therein), wherein another materialsuch as a sponge-like material or hydrophilic polymer is used to keepthe water in place. Such collection reservoirs can be in the form of ahydrogel (for example, in the shape of a disk or pad). Other suitablecollection reservoirs include, but are not limited to, tubes, vials,capillary collection devices, cannulas, and miniaturized etched, ablatedor molded flow paths.

An “ionically conductive material” refers to any material that providesionic conductivity, and through which electrochemically active speciescan diffuse. The ionically conductive material can be, for example, asolid, liquid, or semi-solid (e.g., in the form of a gel) material thatcontains an electrolyte, which can be composed primarily of water andions (e.g., sodium chloride), and generally comprises 50% or more waterby weight. The material can be in the form of a gel, a sponge or pad(e.g., soaked with an electrolytic solution), or any other material thatcan contain an electrolyte and allow passage therethrough ofelectrochemically active species, especially the analyte of interest.

The term “physiological effect” encompasses effects produced in thesubject that achieve the intended purpose of a therapy. In preferredembodiments, a physiological effect means that the symptoms of thesubject being treated are prevented or alleviated. For example, aphysiological effect would be one that results in the prolongation ofsurvival in a patient.

By the term “printed” as used herein is meant a substantially uniformdeposition of an electrode formulation onto one surface of a substrate(i.e., the base support). It will be appreciated by those skilled in theart that a variety of techniques may be used to effect substantiallyuniform deposition of a material onto a substrate, e.g., Gravure-typeprinting, extrusion coating, screen coating, spraying, painting, or thelike.

General Methods

A method and apparatus for sampling small amounts of an analyte viatransdermal methods are provided. The method and apparatus are used todetect and/or quantify the concentration of a target analyte present ina biological system. This sampling is carried out in a continual manner,and quantification is possible even when the target analyte is extractedsample in sub-millimolar concentrations. Although the method andapparatus are broadly applicable to sampling any chemical analyte and/orsubstance, the invention is expressly exemplified for use in transdermalsampling and quantifying or qualifying glucose or a glucose metabolite.

Accordingly, in one aspect of the method of the invention, an automaticsampling system is used to monitor levels of glucose in a biologicalsystem. The method can be practiced using a sampling system (device)which transdermally extracts glucose from the system, in this case, ananimal subject. Transdermal extraction is carried out by applying anelectrical current or ultrasonic radiation to a tissue surface at acollection site. The electrical current or ultrasonic radiation is usedto extract small amounts of glucose from the subject into a collectionreservoir. The collection reservoir is in contact with a biosensor whichprovides for measurement of glucose concentration in the subject.

In the practice of the method, a collection reservoir is contacted witha tissue surface, for example, on the stratum corneum of a patient'sskin. An electrical or ultrasonic force is then applied to the tissuesurface in order to extract glucose from the tissue into the collectionreservoir. Extraction is carried out continually over a period of about1-24 hours, or longer. The collection reservoir is analyzed, at leastperiodically, to measure glucose concentration therein. The measuredvalue correlates with the subject's blood glucose level.

More particularly, one or more collection reservoirs are placed incontact with a tissue surface on a subject. The collection reservoirsare also contacted with an electrode which generates a current (forreverse iontophoretic extraction) or with a source of ultrasonicradiation such as a transducer (for sonophoretic extraction) sufficientto extract glucose from the tissue into the collection reservoir.

The collection reservoir contains an ionically conductive liquid orliquid-containing medium. The conductive medium is preferably a hydrogelwhich can contain ionic substances in an amount sufficient to producehigh ionic conductivity. The hydrogel is formed from a solid material(solute) which, when combined with water, forms a gel by the formationof a structure which holds water including interconnected cells and/ornetwork structure formed by the solute. The solute may be a naturallyoccurring material such as the solute of natural gelatin which includesa mixture of proteins obtained by the hydrolysis of collagen by boilingskin, ligaments, tendons and the like. However, the solute or gelforming material is more preferably a polymer material (including, butnot limited to, polyethylene oxide, polyvinyl alcohol, polyacrylic acid,polyacrylamidomethylpropanesulfonate and copolymers thereof, andpolyvinyl pyrrolidone) present in an amount in the range of more than0.5% and less than 40% by weight, preferably 8 to 12% by weight when ahumectant is also added, and preferably about 15 to 20% by weight whenno humectant is added. Additional materials may be added to thehydrogel, including, without limitation, electrolyte (e.g., a salt),buffer, tackifier, humectant, biocides, preservatives and enzymestabilizers. Suitable hydrogel formulations are described inInternational Publication Nos. WO 97/02811, published Jan. 30, 1997, andWO 96/00110, published Jan. 4, 1996, each of which publications areincorporated herein by reference in their entireties.

Since the sampling system of the present invention must be operated atvery low (electrochemical) background noise levels, the collectionreservoir must contain an ionically conductive medium that does notinclude significant electrochemically sensitive components and/orcontaminants. Thus, the preferred hydrogel composition describedhereinabove is formulated using a judicious selection of materials andreagents which do not add significant amounts of electrochemicalcontaminants to the final composition.

In order to facilitate detection of the analyte, an enzyme is disposedwithin the one or more collection reservoirs. The enzyme is capable ofcatalyzing a reaction with the extracted analyte (in this case glucose)to the extent that a product of this reaction can be sensed, e.g., canbe detected electrochemically from the generation of a current whichcurrent is detectable and proportional to the amount of the analytewhich is reacted. A suitable enzyme is glucose oxidase which oxidizesglucose to gluconic acid and hydrogen peroxide. The subsequent detectionof hydrogen peroxide on an appropriate biosensor electrode generates twoelectrons per hydrogen peroxide molecule which create a current whichcan be detected and related to the amount of glucose entering the device(see FIG. 1). Glucose oxidase (GOx) is readily available commerciallyand has well known catalytic characteristics. However, other enzymes canalso be used, so long as they specifically catalyze a reaction with ananalyte or substance of interest to generate a detectable product inproportion to the amount of analyte so reacted.

In like manner, a number of other analyte-specific enzyme systems can beused in the invention, which enzyme systems operate on much the samegeneral techniques. For example, a biosensor electrode that detectshydrogen peroxide can be used to detect ethanol using an alcohol oxidaseenzyme system, or similarly uric acid with urate oxidase system,cholesterol with a cholesterol oxidase system, and theophylline with axanthine oxidase system.

The biosensor electrode must be able to detect the glucose analyteextracted into the one or more collection reservoirs even when presentat nominal concentration levels. In this regard, conventionalelectrochemical detection systems which utilize glucose oxidase (GOx) tospecifically convert glucose to hydrogen peroxide, and then detect withan appropriate electrode, are only capable of detecting the analyte whenpresent in a sample in at least mM concentrations. In contrast, thepresent invention allows sampling and detection of small amounts ofanalyte from the subject, wherein the analyte is detected atconcentrations on the order of 2 to 4 orders of magnitude lower (e.g.,μM concentration in the reservoir) than presently detectable withconventional systems.

Accordingly, the biosensor electrode of the present invention mustexhibit substantially reduced background current relative to prior suchelectrodes. In one particularly preferred embodiment, an electrode isprovided which contains platinum (Pt) and graphite dispersed within apolymer matrix. The electrode exhibits the following features each ofwhich are essential to the effective operation of the biosensor:background current in the electrode due to changes in the Pt oxidationstate and electrochemically sensitive contaminants in the electrodeformulation is substantially reduced; and catalytic activity (e.g.,nonelectrochemical hydrogen peroxide decomposition) by the Pt in theelectrode is reduced.

The Pt-containing electrode is configured to provide a geometric surfacearea of about 0.1 to 3 cm², preferably about 0.5 to 2 cm², and morepreferably about 1 cm². This particular configuration is scaled inproportion to the collection area of the collection reservoir used inthe sampling system of the present invention, throughout which theextracted analyte and/or its reaction products will be present. Theelectrode is specially formulated to provide a high signal-to-noiseratio (S/N ratio) for this geometric surface area not heretoforeavailable with prior Pt-containing electrodes. For example, aPt-containing electrode constructed according to the invention andhaving a geometric area of about 1 cm² preferably has a backgroundcurrent on the order of about 20 nA or less (when measured with buffersolution at 0.6V), and has high sensitivity (e.g., at least about 60nA/μM of H₂O₂ in buffer at 0.6V). In like manner, an electrode having ageometric area of about 0.1 cm² preferably has a background current ofabout 2 nA or less and sensitivity of at least about 6 nA/μM of H₂O₂;and an electrode having a geometric area of about 3 cm² preferably has abackground current of about 60 nA or less and sensitivity of at leastabout 180 nA/μM of H₂O₂, both as measured in buffer at 0.6V. Thesefeatures provide for a high S/N ratio, for example a S/N ratio of about3 or greater. The electrode composition is formulated using analytical-or electronic-grade reagents and solvents which ensure thatelectrochemical and/or other residual contaminants are avoided in thefinal composition, significantly reducing the background noise inherentin the resultant electrode. In particular, the reagents and solventsused in the formulation of the electrode are selected so as to besubstantially free of electrochemically active contaminants (e.g.,anti-oxidants), and the solvents in particular are selected for highvolatility in order to reduce washing and cure times.

The Pt powder used to formulate the electrode composition is alsosubstantially free from impurities, and the Pt/graphite powders areevenly distributed within the polymer matrix using, for example,co-milling or sequential milling of the Pt and graphite. Alternatively,prior to incorporation into the polymer matrix, the Pt can be sputteredonto the graphite powder, colloidal Pt can be precipitated onto thegraphite powder (see, e.g., U.K. patent application number GB 2,221,300,published Jan. 31, 1990, and references cited therein), or the Pt can beadsorbed onto the graphite powder to provide an even distribution of Ptin contact with the conductive graphite. In order to improve the S/Nratio of the electrode, the Pt content in the electrode is lowerrelative to prior Pt or Pt-based electrodes. In a preferred embodiment,the overall Pt content is about 3-7% by weight. Although decreasing theoverall amount of Pt may reduce the sensitivity of the electrode, theinventors have found that an even greater reduction in background noiseis also achieved, resulting in a net improvement in signal-to-noisequality.

The Pt/graphite matrix is supported by a suitable binder, such as anelectrochemically inert polymer or resin binder, which is selected forgood adhesion and suitable coating integrity. The binder is alsoselected for high purity, and for absence of components withelectrochemical background. In this manner, no electrochemicallysensitive contaminants are introduced into the electrode composition byway of the binder. A large number of suitable such binders are known inthe art and are commercially available, including, without limitation,vinyl, acrylic, epoxy, phenoxy and polyester polymers, provided that thebinder or binders selected for the formulation are adequately free ofelectroactive impurities.

The Pt/graphite biosensor electrodes formulated above exhibit reducedcatalytic activity (e.g., passive or nonelectrochemical hydrogenperoxide degradation) relative to prior Pt-based electrode systems, andthus have substantially improved signal-to-noise quality. In preferredPt/graphite electrode compositions, the biosensor exhibits about 10-25%passive hydrogen peroxide degradation as measured in the assay ofExample 2, infra, preferably less than about 20% passive degradation.

Once formulated, the electrode composition is affixed to a suitablenonconductive surface which may be any rigid or flexible material havingappropriate insulating and/or dielectric properties. The electrodecomposition can be affixed to the surface in any suitable pattern orgeometry, and can be applied using various thin film techniques, such assputtering, evaporation, vapor phase deposition, or the like; or usingvarious thick film techniques, such as film laminating, electroplating,or the like. Alternatively, the composition can be applied using screenprinting, pad printing, inkjet methods, transfer roll printing, orsimilar techniques. Preferably, the electrode is applied using a lowtemperature screen print onto a polymeric substrate. The screening canbe carried out using a suitable mesh, ranging from about 100-400 mesh.

As glucose is transdermally extracted into the collection reservoir, theanalyte reacts with the glucose oxidase within the reservoir to producehydrogen peroxide. The presence of hydrogen peroxide generates a currentat the biosensor electrode that is directly proportional to the amountof hydrogen. peroxide in the reservoir. This current provides a signalwhich can be detected and interpreted (for example, employing analgorithm using statistical methods) by an associated system controllerto provide a glucose concentration value for display.

In one embodiment of the present invention, the sampling system can havetwo collection reservoirs which contain, for example, an activecollection reservoir, having the GOx enzyme, and a blank collectionreservoir (without the GOx enzyme); or, in an alternative, two activereservoirs, i.e., two reservoirs containing the GOx enzyme. In the caseof an active collection reservoir and a blank collection reservoirsignal can be adjusted by subtraction of the blank reservoir signal fromthe signal obtained from the active reservoir. In the case of two activecollection reservoirs the signals can be summed and averaged, or a totalof the two signals can be used. This signal, for example the detectedcurrent, is then used alone or in combination with other factors (forexample, glucose concentration at a calibration point, skin temperature,conductivity, voltage, time since calibration of the system, etc.) toprovide a glucose concentration value.

In particular embodiments, the detected current can be correlated withthe subject's blood glucose concentration (typically using statisticalalgorithms associated with a microprocessor) so that the systemcontroller may display the subject's actual blood glucose concentrationas measured by the sampling system. For example, the system can becalibrated to the subject's actual blood glucose concentration bysampling the subject's blood during a standard glucose tolerance test,and analyzing the blood glucose using both a standard blood glucosemonitor and the sampling system of the present invention. In addition oralternately, the sampling system can be calibrated at a calibration timepoint where the signal obtained from the sampling system at that timepoint is correlated to blood glucose concentration at that time point asdetermined by direct blood testing (for example, glucose concentrationcan be determined using a HemoCue® clinical analyzer (HemoCue AB,Sweden)). In this manner, measurements obtained by the sampling systemcan be correlated to actual values using known statistical techniques.Such statistical techniques can be formulated as algorithm(s) andincorporated in a microprocessor associated with the sampling system.

Further, the sampling system can be preprogrammed to begin execution ofits signal measurements (or other functions) at a designated time. Oneapplication of this feature is to have the sampling system in contactwith a subject and to program the sampling system to begin sequenceexecution during the night so that it is available for calibrationimmediately upon waking. One advantage of this feature is that itremoves any need to wait for the sampling system to warm-up beforecalibrating it.

In one preferred embodiment, the automatic sampling system transdermallyextracts the sample in a continual manner over the course of a 1-24 hourperiod, or longer, using reverse iontophoresis. More particularly, thecollection reservoir contains an ionically conductive medium, preferablythe hydrogel medium described hereinabove. A first iontophoresiselectrode is contacted with the collection reservoir (which is incontact with a target tissue surface), and a second iontophoresiselectrode is contacted with either a second collection reservoir incontact with the tissue surface, or some other ionically conductivemedium in contact with the tissue. A power source provides an electricpotential between the two electrodes to perform reverse iontophoresis ina manner known in the art. As discussed above, the biosensor selected todetect the presence, and possibly the level, of the target analyte(glucose) within a reservoir is also in contact with the reservoir.

In practice, an electric potential (either direct current or a morecomplex waveform) is applied between the two iontophoresis electrodessuch that current flows from the first electrode through the firstconductive medium into the skin, and back out from the skin through thesecond conductive medium to the second electrode. This current flowextracts substances through the skin into the one or more collectionreservoirs through the process of reverse iontophoresis orelectroosmosis. The electric potential may be applied as described inInternational Publication No. WO 96/00110, published Jan. 4, 1996.

As an example, to extract glucose, the applied electrical currentdensity on the skin or tissue is preferably in the range of about 0.01to about 2 mA/cm². In a preferred embodiment, in order to facilitate theextraction of glucose, electrical energy is applied to the electrodes,and the polarity of the electrodes is alternated at a rate of about oneswitch every 7.5 minutes (for a 15 minute extraction period), so thateach electrode is alternately a cathode or an anode. The polarityswitching can be manual or automatic.

Any suitable iontophoretic electrode system can be employed, however itis preferred that a silver/silver chloride (Ag/AgCl) electrode system isused. The iontophoretic electrodes are formulated using two criticalperformance parameters: (1) the electrodes are capable of continualoperation for extended periods, preferably periods of up to 24 hours orlonger; and (2) the electrodes are formulated to have highelectrochemical purity in order to operate within the present systemwhich requires extremely low background noise levels. The electrodesmust also be capable of passing a large amount of charge over the lifeof the electrodes.

In an alternative embodiment, the sampling device can operate in analternating polarity mode necessitating the presence of a first andsecond bimodal electrodes (FIG. 4, 40 and 41) and two collectionreservoirs (FIG. 4, 47 and 48). In the present invention, each bi-modalelectrode (FIG. 3, 30; FIG. 4, 40 and 41) serves two functions dependingon the phase of the operation: (1) an electro-osmotic electrode (oriontophoretic electrode) used to electrically draw analyte from a sourceinto a collection reservoir comprising water and an electrolyte, and tothe area of the electrode subassembly; and (2) as a counter electrode tothe first sensing electrode at which the chemical compound iscatalytically converted at the face of the sensing electrode to producean electrical signal.

The reference (FIG. 4, 44 and 45; FIG. 3, 32) and sensing electrodes(FIG. 4, 42 and 43; FIG. 3, 31), as well as, the bimodal electrode (FIG.4, 40 and 41; FIG. 3, 30) are connected to a standard potentiostatcircuit during sensing. In general, practical limitations of the systemrequire that the bimodal electrode will not act as both a counter andiontophoretic electrode simultaneously.

The general operation of an iontophoretic sampling system is thecyclical repetition of two phases: (1) a reverse-iontophoretic phase,followed by a (2) sensing phase. During the reverse iontophoretic phase,the first bimodal electrode (FIG. 4, 40) acts as an iontophoreticcathode and the second bimodal electrode (FIG. 4, 41) acts as aniontophoretic anode to complete the circuit. Analyte is collected in thereservoirs, for example, a hydrogel (FIG. 4, 47 and 48). At the end ofthe reverse iontophoretic phase, the iontophoretic current is turnedoff. During the sensing phase, in the case-of glucose, a potential isapplied between the reference electrode (FIG. 4, 44) and the sensingelectrode (FIG. 4, 42). The chemical signal reacts catalytically on thecatalytic face of the first-sensing electrode (FIG. 4, 42) producing anelectrical current, while the first bi-modal electrode (FIG. 4, 40) actsas a counter electrode to complete the electrical circuit.

The electrode described is particularly adapted for use in conjunctionwith a hydrogel collection reservoir system for monitoring glucoselevels in a subject through the reaction of collected glucose with theenzyme glucose oxidase present in the hydrogel matrix.

The bi-modal electrode is preferably comprised of Ag/AgCl. Theelectrochemical reaction which occurs at the surface of this electrodeserves as a facile source or sink for electrical current. This propertyis especially important for the iontophoresis function of the electrode.Lacking this reaction, the iontophoresis current could cause thehydrolysis of water to occur at the iontophoresis electrodes causing pHchanges and possible gas bubble formation. The pH changes to acidic orbasic pH could cause skin irritation or burns. The ability of an Ag/AgClelectrode to easily act as a source of sink current is also an advantagefor its counter electrode function. For a three electrodeelectrochemical cell to function properly, the current generationcapacity of the counter electrode must not limit the speed of thereaction at the sensing electrode. In the case of a large sensingelectrode, the ability of the counter electrode to sourceproportionately larger currents is required.

The design of the present invention provides for a larger sensingelectrode (see for example, FIG. 3) than previously designed.Consequently, the size of the bimodal electrode must be sufficient sothat when acting as a counter electrode with respect to the sensingelectrode the counter electrode does not become limiting the rate ofcatalytic reaction at the sensing electrode catalytic surface.

Two methods exist to ensure that the counter electrode does not limitthe current at the sensing electrode: (1) the bi-modal electrode is mademuch larger than the sensing electrode, or (2) a facile counter reactionis provided.

During the reverse iontophoretic phase, the power source provides acurrent flow to the first bi-modal electrode to facilitate theextraction of the chemical signal into the reservoir. During the sensingphase, the power source is used to provide voltage to the first sensingelectrode to drive the conversion of chemical signal retained inreservoir to electrical signal at the catalytic face of the sensingelectrode. The power source also maintains a fixed potential at theelectrode where, for example hydrogen peroxide is converted to molecularoxygen, hydrogen ions, and electrons, which is compared with thepotential of the reference electrode during the sensing phase. While onesensing electrode is operating in the sensing mode it is electricallyconnected to the adjacent bimodal electrode which acts as a counterelectrode at which electrons generated at the sensing electrode areconsumed.

The electrode sub-assembly can be operated by electrically connectingthe bimodal electrodes such that each electrode is capable offunctioning as both an iontophoretic electrode and counter electrodealong with appropriate sensing electrode(s) and reference electrode(s),to create standard potentiostat circuitry.

A potentiostat is an electrical circuit used in electrochemicalmeasurements in three electrode electrochemical cells. A potential isapplied between the reference electrode and the sensing electrode. Thecurrent generated at the sensing electrode flows through circuitry tothe counter electrode (i.e., no current flows through the referenceelectrode to alter its equilibrium potential). Two independentpotentiostat circuits can be used to operate the two biosensors. For thepurpose of the present invention, the electrical current measured at thesensing electrode subassembly is the current that is correlated with anamount of chemical signal.

With regard to continual operation for extended periods of time, Ag/AgClelectrodes are provided herein which are capable of repeatedly forming areversible couple which operates without unwanted electrochemical sidereactions (which could give rise to changes in pH, and liberation ofhydrogen and oxygen due to water hydrolysis). The Ag/AgCl electrodes ofthe present invention are thus formulated to withstand repeated cyclesof current passage in the range of about 0.01 to 1.0 mA per cm² ofelectrode area. With regard to high electrochemical purity, the Ag/AgClcomponents are dispersed within a suitable polymer binder to provide anelectrode composition which is not susceptible to attack (e.g.,plasticization) by components in the collection reservoir, e.g., thehydrogel composition. The electrode compositions are also formulatedusing analytical- or electronic-grade reagents and solvents, and thepolymer binder composition is selected to be free of electrochemicallyactive contaminants which could diffuse to the biosensor to produce abackground current.

Since the Ag/AgCl iontophoretic electrodes must be capable of continualcycling over extended periods of time, the absolute amounts of Ag andAgCl available in the electrodes, and the overall Ag/AgCl availabilityratio, can be adjusted to provide for the passage of high amounts ofcharge. Although not limiting in the present invention, the Ag/AgClratio can approach unity. In order to operate within the preferredsystem which uses a biosensor having a geometric area of 0.1 to 3 cm²,the iontophoretic electrodes are configured to provide an approximateelectrode area of 0.3 to 1.0 cm², preferably about 0.85 cm². Theseelectrodes provide for reproducible, repeated cycles of charge passageat current densities ranging from about 0.01 to 1.0 mA/cm² of electrodearea. More particularly, electrodes constructed according to the aboveformulation parameters, and having an approximate electrode area of 0.85cm², are capable of a reproducible total charge passage (in both anodicand cathodic directions) of 270 mC, at a current of about 0.3 mA(current density of 0.35 mA/cm²) for 48 cycles in a 24 hour period.

Once formulated, the Ag/AgCl electrode composition is affixed to asuitable rigid or flexible nonconductive surface as described above withrespect to the biosensor electrode composition. A silver (Ag) underlayeris first applied to the surface in order to provide uniform conduction.The Ag/AgCl electrode composition is then applied over the Ag underlayerin any suitable pattern or geometry using various thin film techniques,such as sputtering, evaporation, vapor phase deposition, or the like, orusing various thick film techniques, such as film laminating,electroplating, or the like. Alternatively, the Ag/AgCl composition canbe applied using screen printing, pad printing, inkjet methods, transferroll printing, or similar techniques. Preferably, both the Ag underlayerand the Ag/AgCl electrode are applied using a low temperature screenprint onto a polymeric substrate. This low temperature screen print canbe carried out at about 125 to 160° C., and the screening can be carriedout using a suitable mesh, ranging from about 100-400 mesh.

In another preferred embodiment, the automatic sampling systemtransdermally extracts the sample in a continual manner over the courseof a 1-24 hour period, or longer, using sonophoresis. More particularly,a source of ultrasonic radiation is coupled to the collection reservoirand used to provide sufficient perturbation of the target tissue surfaceto allow passage of the analyte (glucose) across the tissue surface. Thesource of ultrasonic radiation provides ultrasound frequencies ofgreater than about 10 MHz, preferably in the range of about 10 to 50MHz, most preferably in the range of about 15 to 25 MHz. It should beemphasized that these ranges are intended to be merely illustrative ofthe preferred embodiment; in some cases higher or lower frequencies maybe used.

The ultrasound may be pulsed or continuous, but is preferably continuouswhen lower frequencies are used. At very high frequencies, pulsedapplication will generally be preferred so as to enable dissipation ofgenerated heat. The preferred intensity of the applied ultrasound isless than about 5.0 W/cm², more preferably is in the range of about 0.01to 5.0 W/cm² , and most preferably is in the range of 0.05 to 3.0 W/cm².

Virtually any type of device may be used to administer the ultrasound,providing that the device is capable of producing the suitable frequencyultrasonic waves required by the invention. An ultrasound device willtypically have a power source such as a small battery, a transducer, anda means to attach the system to the sampling system collectionreservoir. Suitable sonophoresis sampling systems are described inInternational Publication No. WO 91/12772, published Sep. 5, 1991, thedisclosure of which is incorporated herein by reference.

As ultrasound does not transmit well in air, a liquid medium isgenerally needed in the collection reservoir to efficiently and rapidlytransmit ultrasound between the ultrasound applicator and the tissuesurface.

Referring now to FIG. 2, an exploded view of the key components from apreferred embodiment of an iontophoretic sampling system is presented.The sampling system components include two biosensor/iontophoreticelectrode assemblies, 4 and 6, each of which have an annulariontophoretic electrode, respectively indicated at 8 and 10, whichencircles a biosensor 12 and 14. The electrode assemblies 4 and 6 areprinted onto a polymeric substrate 16 which is maintained within asensor tray 18. A collection reservoir assembly 20 is arranged over theelectrode assemblies, wherein the collection reservoir assemblycomprises two hydrogel inserts 22 and 24 retained by a gel retainer 26.

In one embodiment, the electrode assemblies can include bimodalelectrodes as shown in FIG. 3 and described above.

The components shown in exploded view in FIG. 2 are intended for use ina automatic sampling system which is configured to be worn like anordinary wristwatch. As described in International Publication No. WO96/00110, published Jan. 4, 1996, the wristwatch housing (not shown)contains conductive leads which communicate with the iontophoreticelectrodes and the biosensor electrodes to control cycling and providepower to the iontophoretic electrodes, and to detect electrochemicalsignals produced at the biosensor electrode surfaces. The wristwatchhousing can further include suitable electronics (e.g., microprocessor,memory, display and other circuit components) and power sources foroperating the automatic sampling system.

Modifications and additions to the embodiment of FIG. 2 will be apparentto those skilled in the art.

It is to be understood that while the invention has been described inconjunction with the preferred specific embodiments thereof, that thedescription above as well as the examples which follow are intended toillustrate and not limit the scope of the invention. Other aspects,advantages and modifications within the scope of the invention will beapparent to those skilled in the art to which the invention pertains.

In the following examples, efforts have been made to ensure accuracywith respect to numbers used (e.g., amounts, temperature, etc.) but someexperimental error and deviation should be accounted for. Unlessindicated otherwise, temperature is in degrees C and pressure is at ornear atmospheric.

All patents, patent applications, and publications mentioned herein,both supra and infra, are hereby incorporated by reference.

EXAMPLE 1 Assessing Background Noise and Sensitivity in a Biosensor

The following procedure can be used to determine the background currentand sensitivity to hydrogen peroxide of a biosensor electrode in aglucose monitoring system.

Method: The sensitivity and background of the working biosensorelectrode is determined by placing the biosensor in a test cell of fixedvolume. The cell is filled with a pH 7.5 0.1 M phosphate buffered saline(PBS) solution containing 77 mM NaCl. The buffer solution is quiescentin the cell during measurement. The biosensor is then biased at theusual operating potential of 0.6V, and the steady-state backgroundcurrent measured. A 2 μM solution of hydrogen peroxide is then preparedin the same PBS buffer solution, and is added to the test cell. Thebiosensor is again biased, and the current measured at fixed timepoints. The measurement is repeated for 5 μM and 10 μM hydrogen peroxidesolutions.

A calibration curve can be constructed from the currents obtained forthe three hydrogen peroxide concentrations for the fixed time points.Because the currents decay over time after the sensor bias is applied,it is best to pick one standard time point (for example 60 seconds) tocharacterize the sensitivity of the biosensor to hydrogen peroxide.

The hydrogen peroxide solution should be made up within 2 hours of thetest, and stored in an amber (or foil-covered) container until use toprevent decomposition of the hydrogen peroxide.

The following ingredients can be used to make up the PBS buffer (NaCl,NaH₂PO₄.H₂O, and Na₂HPO₄.7H₂O). The recipe to make up this pH 7.5 bufferis as follows:

To make 2 liters of 0.05 M pH 7.5 phosphate-buffered saline (PBS): 2.20g NaH₂PO₄.H₂O+22.5 g Na₂HPO₄.7H₂O+8.98 g NaCl; add water to bring to 2liters. The hydrogen peroxide solutions can be made from 3% stocksolution which is stable if stored in the refrigerator.

EXAMPLE 2 Assessing Passive Hydrogen Peroxide Depletion in a Biosensor

The following procedure can be used to determine the rate of passive(non-electrochemical) hydrogen peroxide depletion caused by a biosensorelectrode constructed in accordance with the present invention.

Method: The following procedure is optimized to test biosensorelectrodes having an approximate geometric area of 1 cm², and a totalbiosensor area of about 3 cm²; however, these methods are readilyscalable to smaller or larger electrode dimensions by the ordinarilyskilled artisan.

The biosensor to be tested is placed in a test cell which contains avolume of test solution of approximately 360 μL in contact with theelectrode. The thickness of the solution layer in contact with thebiosensor electrode is approximately 50 mil (0.127 cm). The body of thetest cell is preferably made from materials that do not cause asubstantial amount of hydrogen peroxide degradation, for examplepolytetrafluoroethylene (e.g., TEFLON®) or poly(chlorotrifluoroethylene)(e.g., Kel-F®). The test solution contains 20 μM hydrogen peroxide in0.1 M PBS (pH 7.5) containing NaCl. The test solution is added to thecell and left in contact with the biosensor for 15 minutes. The testsolution is withdrawn from the cell, and the remaining concentration ofhydrogen peroxide is measured and compared to standard solutions thatwere not contacted with the biosensor. The concentration of hydrogenperoxide remaining in the test solution can then determined usingseveral methods known to those of skill in the art, for example, byliquid chromatography or by one of several commercially availablehydrogen peroxide test kits (e.g., PeroXOquant™, available from PierceChemical Co., Rockford Ill.).

Solid Pt biosensor electrodes tested in the above-describedprocedure.generally exhibit from 55-75% passive (non-electrochemical)hydrogen peroxide degradation, whereas Pt-containing electrodesconstructed according to the invention preferably exhibit from about10-25% passive hydrogen peroxide degradation, and more preferably lessthan about 20% passive degradation.

EXAMPLE 3 Testing Protocol for Ag/AqCl Screen-Printed Electrodes

The following procedure can be used determine the amount of Ag and AgClin a screen printed Ag/AgCl electrode that is electrochemicallyaccessible at a given current density.

Method: The amount of available Ag and AgCl is determined by passing aconstant current between the Ag/AgCl electrode and a counter electrodeimmersed in a suitable electrolyte until an increase in the appliedvoltage indicates that depletion of Ag or AgCl has occurred.

Test of available Ag: The Ag/AgCl electrode and a much larger counterelectrode of chloridized silver foil are placed opposite each other in abeaker. The beaker is filled with 0.1 M PBS buffer (pH 7.5) containing77 mM NaCl. The electrodes are connected to a suitable constant currentpower source and the electrodes biased so that the Ag/AgCl electrode ispositive with respect to the counter electrode. A constant currentpasses between the electrodes. The applied potential is monitored andthe amount of time required for the applied potential to reach 1.0 V ismeasured. (At 1.0 V, undesirable side reactions can start occurring.)The amount of charge which is passed is equal to the current times thenumber of seconds.

Test of available AgCl: The Ag/AgCl electrode and a much larger counterelectrode of chloridized silver foil are placed opposite each other in abeaker. The beaker is filled with 0.1 M PBS buffer (pH 7.5) containing77 mM NaCl. The electrodes are connected to a suitable constant currentpower source and the electrodes biased so that the Ag/AgCl electrode isnegative with respect to the counter electrode. A constant currentpasses between the electrodes. The applied potential is monitored andthe amount of time required for the applied potential to reach −1.0 V ismeasured. (At −1.0 V, undesirable side reactions can start occurring.)The amount of charge which is passed is equal to the current times thenumber of seconds.

A preferred Ag/AgCl iontophoresis electrode for use in the presentinvention to provide for a 24 hour period of continual sampling can havethe following specifications: (1) an electrode area of about 0.85 cm²;(2) current of 0.3 mA (current density: 0.35 mA/cm²); (3) available Agand AgCl requirement per electrode of 0.5 millicoulomb/cm² @ 0.9 mA; (4)time duration of current: 7.5 minutes in each direction per cycle; (5)total charge passage of 135 mC (in each of the anodic and cathodicdirections for a total of 270 mC); and (6) capable of at least 48anode/cathode cycles.

What is claimed is:
 1. A sampling system for monitoring theconcentration or amount of an analyte present in a biological system,said sampling system comprising: (a) a collection reservoir comprisingan enzyme capable of reacting with the analyte to produce hydrogenperoxide; (b) sampling means for extracting the analyte from thebiological system into the collection reservoir to obtain asub-millimolar (sub-mM) concentration or amount of the analyte in thecollection reservoir, wherein said sampling means is in operativecontact with the collection reservoir; and (c) a sensor element inoperative contact with the collection reservoir, said sensor elementcomprising an electrode, wherein said electrode reacts electrochemicallywith the hydrogen peroxide produced in the collection reservoir toprovide a detectable signal, said electrode comprising a geometricsurface area that ranges from about 0.1 to 3 cm², a background currentthat ranges from about 2 to 60 nA or less when measured in a buffersolution at 0.6V, and a sensitivity that ranges from about 6 to 180nA/μM of hydrogen peroxide when measured in a buffer solution at 0.6V.2. The sampling system of claim 1, wherein the sensor element comprisesa platinum-group metal-containing electrode.
 3. The sampling system ofclaim 2, wherein the platinum-group metal is platinum.
 4. The samplingsystem of claim 3, wherein the electrode comprises about 3-7% by weightof platinum dispersed in a polymer matrix.
 5. The sampling system ofclaim 1, wherein the sampling means comprises first and secondcollection reservoirs, wherein each collection reservoir comprises anionically conductive medium and at least one collection reservoirfurther comprises an enzyme capable of reacting with the analyte toproduce hydrogen peroxide and said sampling means uses reverseiontophoresis to extract the analyte from the biological system.
 6. Thesampling system of claim 5, wherein the sampling means comprises aniontophoretic electrode comprising a geometric area ranging from about0.3 to 1.0 cm², and capable of repeated cycles of current passage in therange of about 0.01 to 1.0 mA/cm² of electrode area.
 7. The samplingsystem of claim 7, wherein the iontophoretic electrode is a bimodalelectrode that is also able to act as a counter electrode, and whereinthe sensor element comprises a sensing electrode and a referenceelectrode.
 8. The sampling system of claim 1, wherein the sensor elementcomprises a sensing electrode, a counter electrode, and a referenceelectrode.
 9. The sampling system of claim 1, wherein the sampling meansto extract the analyte from the biological system is selected from thegroup consisting of sonophoresis, suction, electroporation, thermalporation, passive diffusion, microfine lances, microfine cannulas,subcutaneous implants, subcutaneous insertions, microdialysis, andlaser.
 10. The sampling system of claim 9, wherein the sampling means toextract the analyte from the biological system is selected from thegroup consisting of sonophoresis, suction, electroporation, thermalporation, passive diffusion, subcutaneous implants, subcutaneousinsertions, microdialysis, and laser.
 11. The sampling system of claim10, wherein (i) said biological system comprises a skin surface, and(ii) said sampling system further comprises a skin permeabilityenhancer, said skin permeability enhancer comprising micro-needles. 12.The sampling system of claim 1, wherein the analyte comprises glucose,and the enzyme comprises glucose oxidase.
 13. The sampling system ofclaim 1, wherein said collection reservoir further comprises anionically conductive medium.
 14. The sampling system of claim 13,wherein the ionically conductive medium comprises a hydrogel comprisingsaid enzyme capable of reacting with the analyte to produce hydrogenperoxide.
 15. The sampling system of claim 1, wherein the electrodecomprises a geometric area of about 1 cm², a background current of about20 nA, and a sensitivity of about 60 nA/μM of hydrogen peroxide.
 16. Thesampling system of claim 1, wherein the electrode is printed onto arigid or flexible substrate.
 17. The sampling system of claim 1, whereinthe electrode exhibits passive hydrogen peroxide depletion in the rangeof about 25%.
 18. The sampling system of 1, wherein (i) said biologicalsystem comprises a skin surface, and (ii) said sampling system furthercomprises a skin permeability enhancer, said skin permeability enhancercomprising micro-needles.
 19. The sampling system of claim 1, whereinthe sampling means comprises sonophoresis to extract the analyte fromthe biological system.
 20. A sampling system for monitoring theconcentration or amount of an analyte present in a biological system,said sampling system comprising: (a) first and second collectionreservoirs, wherein each collection reservoir comprises an ionicallyconductive medium and at least one collection reservoir furthercomprises an enzyme capable of reacting with the analyte to producehydrogen peroxide; (b) reverse iontophoretic sampling means forextracting the analyte from the biological system into the collectionreservoir to obtain a sub-millimolar (sub-mM) concentration or amount ofthe analyte in the collection reservoir, wherein said sampling means isin operative contact with the collection reservoir and comprises firstand second iontophoretic electrodes in contact with said first andsecond collection reservoirs, wherein each iontophoretic electrodecomprises a geometric area ranging from about 0.3 to 1.0 cm² , and iscapable of repeated cycles of current passage in the range of about 0.01to 1.0 mA/cm² of electrode area; and (c) first and second sensorelements in operative contact with the first and second collectionreservoirs, wherein each sensor element comprises an electrode thatreacts electrochemically with the hydrogen peroxide produced in thecollection reservoir to provide a detectable signal, each electrodecomprising a geometric surface area that ranges from about 0.1 to 3 cm², a background current that ranges from about 2 to 60 nA or less whenmeasured in a buffer solution at 0.6V, and a sensitivity that rangesfrom about 6 to 180 nA/μM of hydrogen peroxide when measured in a buffersolution at 0.6V.
 21. The sampling system of claim 20, wherein theiontophoretic electrodes provide for at least 48 anodic/cathodic cyclesin a 24 hour period.
 22. The sampling system of claim 20, wherein theiontophoretic electrodes comprise silver/silver chloride (Ag/AgCl)dispersed in a polymer binder.
 23. The sampling system of claim 22,wherein the iontophoretic electrodes each have a geometric area of about0.85 cm², and provide a reproducible total charge passage in both anodicand cathodic directions of about 270 mC at a current density of about0.35 mA/cm².
 24. The sampling system of claim 20, wherein the ionicallyconductive medium comprises a hydrogel.
 25. The sampling system of claim20, wherein the iontophoretic electrodes are printed onto a rigid orflexible substrate using a low temperature screen print.
 26. Thesampling system of claim 20, wherein each sensor element comprises asensing electrode, a counter electrode, and a reference electrode. 27.The sampling system of claim 20, herein each iontophoretic electrode isa bimodal electrode that is also able to act as a counter electrode, andeach sensor element comprises a sensing electrode and a referenceelectrode.
 28. The sampling system of claim 20, wherein the analytecomprises glucose, and the enzyme comprises glucose oxidase.
 29. Thesampling system of 20, wherein (i) said biological system comprises askin surface, and (ii) said sampling system further comprises a skinpermeability enhancer, said skin permeability enhancer comprisingmicro-needles.
 30. A method for monitoring the concentration or amountof an analyte present in a biological system, said method comprising:(a) extracting said analyte from the biological system into a collectionreservoir to obtain a sub-millimolar (sub-mM) concentration or amount ofthe analyte in said collection reservoir; (b) contacting the analyteextracted in step (a) with an enzyme that reacts with the analyte toproduce hydrogen peroxide; (c) detecting the hydrogen peroxide producedin step (b) with a sensor element comprising an electrode that reactselectrochemically with the hydrogen peroxide to produce a detectablesignal, said electrode comprising a geometric surface area that rangesfrom about 0.1 to 3 cm², a background current that ranges from about 2to 60 nA or less when measured in a buffer solution at 0.6V, and asensitivity that ranges from about 6 to 180 nA/μM of hydrogen peroxidewhen measured in a buffer solution at 0.6V; (d) measuring the signalproduced in step (c); (e) correlating the measurement obtained in step(d) with the concentration or amount of the analyte in the biologicalsystem; and (f) performing repeated cycles of steps (a)-(e) over a timeperiod to monitor the concentration or amount of the analyte in thebiological system.
 31. The method of claim 30, wherein said time periodis at least about 12 hours.
 32. The method of claim 30, wherein thebiological system is a mammalian subject.
 33. The method of claim 32,wherein said extracting of the analyte comprises extracting the analyteby transdermal extraction.
 34. The method of claim 33, wherein saidextracting of the analyte comprises use of a sampling means comprisingfirst and second collection reservoirs, wherein each collectionreservoir comprises an ionically conductive medium and at least saidfirst collection reservoir further comprises an enzyme capable ofreacting with the analyte to produce hydrogen peroxide and said samplingmeans uses reverse iontophoresis to extract the analyte from thebiological system.
 35. The method of claim 34, wherein said extractingof the analyte comprises using a reverse iontophoresis sampling systemcomprising first and second iontophoretic electrodes each comprising ageometric area ranging from about 0.3 to 1.0 cm², and that are capableof repeated cycles of current passage in the range of about 0.01 to 1.0mA/cm² of electrode area.
 36. The method of claim 35, wherein the sensorelement comprises a sensing electrode, a counter electrode, and areference electrode.
 37. The method of claim 35, wherein at least saidfirst iontophoretic electrode is a bimodal electrode that is also ableto act as a counter electrode, and wherein the sensor element comprisesa sensing electrode and a reference electrode.
 38. The method of claim33, wherein said extracting of the analyte comprises using a samplingmeans selected from the group consisting of sonophoresis, suction,electroporation, thermal poration, passive diffusion, microfine lances,microfine cannulas, subcutaneous implants, subcutaneous insertions,microdialysis, and laser.
 39. The method of claim 33, wherein saidextracting of the analyte comprises sonophoresis.
 40. The method ofclaim 33, wherein said extracting of the analyte comprises using asampling means selected m the group consisting of sonophoresis, suction,electroporation, thermal poration, passive diffusion, subcutaneousimplants, subcutaneous insertions, microdialysis, and laser.
 41. Themethod of claim 40, wherein said biological system comprises a skinsurface and said extracting of analyte from the biological system into acollection reservoir further comprises enhancement of skin permeability.42. The method of claim 30, wherein said time period is at least about24 hours.
 43. The method of claim 30, wherein the analyte is present inthe biological system at a concentration or amount ranging from about0.1 to 200 millimolar (mM).
 44. The method of claim 30, wherein theanalyte comprises glucose, and the enzyme comprises glucose oxidase. 45.The method of claim 30, wherein the sensor element electrode exhibitsreduced passive hydrogen peroxide depletion in the range of about 25% orless.
 46. The method of claim 30, wherein said biological systemcomprises skin, and said extracting of analyte from the biologicalsystem into a collection reservoir further comprises enhancement of skinpermeability by pricking the skin with micro-needles.
 47. A method formonitoring the concentration or amount of an analyte present in abiological system, said method comprising: (a) contacting a samplingsystem with the biological system, said sampling system comprising (i)first and second collection reservoirs, wherein each collectionreservoir comprises an ionically conductive medium and at least thefirst collection reservoir further comprises an enzyme capable ofreacting with the analyte to produce hydrogen peroxide; (ii) reverseiontophoretic sampling means for extracting the analyte from thebiological system into the first collection reservoir to obtain asub-millimolar (sub-mM) concentration or amount of the analyte in thecollection reservoir, wherein said sampling means comprises first andsecond iontophoretic electrodes in operative contact with said first andsecond collection reservoirs, wherein each iontophoretic electrodecomprises a geometric area ranging from about 0.3 to 1.0 cm², and iscapable of repeated cycles of current passage in the range of about 0.01to 1.0 mA/cm² of electrode area; and (iii) first and second sensorelements in operative contact with the first and second collectionreservoirs, wherein each sensor element comprises an electrode capableof reacting electrochemically with the hydrogen peroxide produced in thecollection reservoir to provide a detectable signal, each electrodecomprising a geometric surface area that ranges from about 0.1 to 3 cm²,a background current that ranges from about 2 to 60 nA or less whenmeasured in a buffer solution at 0.6V, and a sensitivity that rangesfrom about 6 to 180 nA/μM of hydrogen peroxide when measured in a buffersolution at 0.6V; (b) extracting said analyte from the biological systeminto the first collection reservoir to obtain a sub-millimolar (sub-mM)concentration or amount of the analyte in the first collectionreservoir, wherein said extracting is carried out using the reverseiontophoretic system; (c) contacting the analyte extracted in step (b)with an enzyme that reacts with the analyte to produce hydrogenperoxide; (d) detecting the hydrogen peroxide produced in step (c) withthe first sensor element that reacts electrochemically with the hydrogenperoxide to produce a detectable signal; (e) measuring the signalproduced in step (d); (f) correlating the measurement obtained in step(e) with the concentration or amount of the analyte in the biologicalsystem; and (g) performing repeated cycles of steps (b)-(f) over a timeperiod to monitor the concentration or amount of the analyte in thebiological system.
 48. The method of claim 47, wherein said time periodis at least about 12 hours.
 49. The method of claim 47, wherein saidtime period is at least about 24 hours.
 50. The method of claim 49,wherein the iontophoretic electrodes provide for at least 48anodic/cathodic cycles in a 24 hour period.
 51. The method of claim 47,wherein the biological system is a mammalian subject.
 52. The method ofclaim 47, wherein the analyte is present in the biological system at aconcentration or amount ranging from about 0.1 to 200 millimolar (mM).53. The method of claim 47, wherein the analyte comprises glucose, andthe enzyme comprises glucose oxidase.
 54. The method of claim 47,wherein at least said first sensor element comprises a sensingelectrode, a counter electrode, and a reference electrode.
 55. Themethod of claim 47, wherein each iontophoretic electrode is a bimodalelectrode that is also able to act as a counter electrode, and whereineach sensor element comprises a sensing electrode and a referenceelectrode.
 56. The method of claim 47, wherein said biological systemcomprises skin, and said extracting of analyte from the biologicalsystem into a collection reservoir further comprises enhancement of skinpermeability by pricking with micro-needles.
 57. A method for monitoringthe concentration or amount of an analyte present in a biologicalsystem, said method comprising: (a) contacting a sampling system withthe biological system, said sampling system comprising (i) first andsecond collection reservoirs, wherein each collection reservoircomprises an ionically conductive medium and an enzyme capable ofreacting with the analyte to produce hydrogen peroxide; (ii) reverseiontophoretic sampling means for extracting the analyte from thebiological system into the collection reservoirs to obtain asub-millimolar (sub-mM) concentration or amount of the analyte in thecollection reservoirs, wherein said sampling means comprises first andsecond iontophoretic electrodes in operative contact with said first andsecond collection reservoirs, wherein each iontophoretic electrodecomprises a geometric area ranging from about 0.3 to 1.0 cm², and iscapable of repeated cycles of current passage in the range of about 0.01to 1.0 mA/cm² of electrode area; and (iii) first and second sensorelements in operative contact with the first and second collectionreservoirs, wherein each sensor element comprises an electrode capableof reacting electrochemically with the hydrogen peroxide produced in thecollection reservoir to provide a detectable signal, each electrodecomprising a geometric surface area that ranges from about 0.1 to 3 cm²,a background current that ranges from about 2 to 60 nA or less whenmeasured in a buffer solution at 0.6V, and a sensitivity that rangesfrom about 6 to 180 nA/μM of hydrogen peroxide when measured in a buffersolution at 0.6V; (b) extracting said analyte from the biological systeminto the first collection reservoir to obtain a sub-millimolar (sub-mM)concentration or amount of the analyte in the first collectionreservoir, wherein said extracting is carried out using the reverseiontophoretic system; (c) contacting the analyte extracted in step (b)with an enzyme that reacts with the analyte to produce hydrogenperoxide; (d) detecting the hydrogen peroxide produced in step (c) withthe first sensor element that reacts electrochemically with the hydrogenperoxide to produce a detectable signal; (e) measuring the signalproduced in step (d); (f) correlating the measurement obtained in step(e) with the concentration or amount of the analyte in the biologicalsystem; (g) extracting said analyte from the biological system into thesecond collection reservoir to obtain a sub-millimolar (sub-mM)concentration or amount of the analyte in the second collectionreservoir, wherein said extracting is carried out using the reverseiontophoretic system; (h) contacting the analyte extracted in step (g)with an enzyme that reacts with the analyte to produce hydrogenperoxide; (i) detecting the hydrogen peroxide produced in step (h) withthe second sensor element that reacts electrochemically with thehydrogen peroxide to produce a detectable signal; (j) measuring thesignal produced in step (i); (k) correlating the measurement obtained instep (j) with the concentration or amount of the analyte in thebiological system; and (l) performing repeated cycles of (i) steps(b)-(e) alternating with (ii) steps (g)-(j) over a time period, andperiodically performing steps (f) and (k) over the time period tomonitor the concentration or amount of the analyte in the biologicalsystem.
 58. The method of claim 57, wherein the biological system is amammalian subject.
 59. The method of claim 57, wherein the analyte ispresent in the biological system at a concentration or amount rangingfrom about 0.1 to 200 millimolar (mM).
 60. The method of claim 57,wherein the analyte comprises glucose, and the enzyme comprises glucoseoxidase.
 61. The method of claim 57, wherein each sensor elementcomprises a sensing electrode, a counter electrode, and a referenceelectrode.
 62. The method of claim 57, wherein each iontophoreticelectrode is a bimodal electrode that is also able to act as a counterelectrode, and wherein each sensor element comprises a sensing electrodeand a reference electrode.
 63. The method of claim 57, wherein saidbiological system comprises skin, and said extracting of analyte fromthe biological system into a collection reservoir further comprisesenhancement of skin permeability by pricking the skin withmicro-needles.